Characterization of the metallo-[beta]-lactamase L1 from Stenotrophomonas maltophilia

by Periyannan, Gopal Raj.

Abstract (Summary)

CHARACTERIZATION OF METALLO-?-LACTAMASE L1 FROM
STENOTROPHOMONAS MALTOPHILIA
by Gopal Raj Periyannan
Zinc-containing metallo-?-lactamases (m?Ls) are an emerging class of enzymes that
render bacteria resistant to ?-lactam-containing antibiotics. In an effort to better understand
the structure and function of the m?L L1 from Stenotrophomonas maltophilia, spectroscopic
characterization of native and Co(II)-substituted L1 was performed. Co(II)-substituted L1
was characterized by using UV-Vis, EPR, EXAFS, and 1H paramagnetic NMR spectroscopic
techniques. Our data show that Co(II) and Zn(II) bind L1 sequentially and preferentially. A
model for Co(II) and Zn(II) binding to L1 is presented. Attempts were made to prepare
mixed-metal analogs (CoZn or ZnCo) of L1, and our data show that only the ZnCo-analog
can be prepared. In the concluding remarks, an ingenious way to use the ZnCo-analog of L1
to probe the reaction mechanism using spectroscopic techniques is provided. Metal-binding
histidine ligands were mutated to cysteines in order to prepare the CoZn analog of L1;
however, the binding preference of Co(II) and Zn(II) prevents the preparation of the CoZn
analog of L1. The Co(II) and Zn(II) binding modes of the subgroup 3a m?L CcrA from
Bacteroides fragilis was also studied using EPR, EXAFS, and 1H NMR spectroscopic
techniques. In contrast to L1, Co(II) and Zn(II) both bind to the 3 His Zn1 site in CcrA first
before binding to the Zn2 site.
To probe metal incorporation in m?L, L1 was over-expressed in a minimal media
with and without Zn(II), and the resulting proteins were purified and characterized by steadystate
kinetics and CD, and fluorescence spectroscopies. Our data demonstrate that the proper
in vivo folding of L1 requires the presence of Zn(II); however, in vitro folding of L1 does not
require Zn(II). Our results suggest that in vitro refolding experiments do not necessarily
reflect in vivo folding conditions.